This invention concerns aspects of a process for the production of ascorbic acid. The invention relates to improvements in the method for converting an ordinary metabolite, such as D-glucose, into 2-keto-L-gulonate (2-KLG), a stable precursor of ascorbic acid. The invention relates to the use of recombinant technology to alter the metabolism of a microorganism to carry out a desired bioconversion. It specifically relates to the use of recombinant technology to transfer genetic material to a microorganism such that transfer renders the cell capable of effecting the conversion of an ordinary metabolite to 2-KLG and to control the effect of secondary metabolites or metabolic pathways on that conversion. More specifically, the invention relates to the elimination and/or modulation of the level of secondary metabolites such as L-idonate or altering the enzymatic activity in a metabolic pathway, such as, for example, altering the activity of 2-ketoaldonate reductase.
Ascorbic acid has become a major chemical product in the United States, and elsewhere in the world, due to its importance in health maintenance. While there may be some controversy over its efficacy in ameliorating the tendency of individuals to contract certain minor illnesses, such as, for example, the common cold, there is no doubt that it is essential for human beings to ingest required amounts of vitamin C. It has become a matter of concern in recent years that "natural" foods may not provide adequate amounts of vitamin C. Accordingly, there has developed a large demand for ascorbic acid, both as an additive to foods which are marketed to the consumer with supplemented levels of this vitamin, and as a direct vitamin supplement. Furthermore, ascorbic acid is an effective antioxidant and thus finds applications as a preservative both in nutritional and in other products.
There are a number of processes available, some commercially viable, for the production of vitamin C. Several of these result in the preliminary production of 2-keto-L-gulonic acid (2-KLG) which can then be rather simply converted to ascorbic acid through acid or base catalyzed cyclization. Accordingly, 2-KLG has become, in itself, a material of considerable economic and industrial importance.
Means are presently available in the art to convert ordinary metabolites, such as, for example, D-glucose, into 2,5-diketo-D-gluconic acid (2,5-DKG) by processes involving the metabolism of prokaryotic microorganisms. See, for example, U.S. Pat. No. 3,790,444 (Feb. 5, 1974); 3,998,697 (Dec. 21, 1976); and EPO Application Publication No. 0046284 published Feb. 24, 1982. The availability of this 2,5-DKG intermediate offers a starting material which is converted to the desired 2-KLG by a single step reduction. The reduction can be effected chemically or catalyzed enzymatically. Various bacterial strains are known which are capable of effecting this reduction. Such strains are found in the genera Brevibacterium, Arthrobacter, Micrococcus, Staphylococcus, Pseudomonas, Bacillus, Citrobacter and Corynebacterium. See, for example, U.S. Pat. No. 3,922,194 (Nov. 25, 1975), U.S. Pat. No. 4,245,049 (Jan. 13, 1981) and U.S. Pat. No. 3,959,076 (May 25, 1976).
It is helpful to understand the context into which the present invention finds utility by representing the process in terms of the relevant chemical conversions. An outline of a typical overall process for manufacture of ascorbic acid is shown in Reaction Scheme 1. ##STR1##